Sensors and Actuators B 253 (2017) 723–730

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Sensors and Actuators B: Chemical

journal homepage: www.elsevier.com/locate/snb

Enhanced peroxidase-like activity of CuWO4 nanoparticles for the

detection of NADH and hydrogen peroxide
K. Aneesh a,b , Chiranjeevi Srinivasa Rao vusa b , Sheela Berchmans a,b,∗
a
Academy of Scientific and Innovative Research (AcSIR), India
b
CSIR-Central Electrochemical Research Institute, Karaikudi 630003, Tamil Nadu, India

a r t i c l e i n f o a b s t r a c t

Article history: Herein, we demonstrate for the first time the peroxidase mimicking properties of CuWO4 nanoparticles
Received 29 March 2017 (CuWO4 NPs) and a simple colorimetric method for the determination of nicotinamide adenine dinucleo-
Received in revised form 21 June 2017 tide (NADH) based on the inhibition of peroxidase-like properties of CuWO4 NPs. A one-pot hydrothermal
Accepted 26 June 2017
process was used to synthesize the CuWO4 NPs and XRD, SEM, HR-TEM and XPS were used to confirm
Available online 28 June 2017
the formation of CuWO4 NPs. The synthesized CuWO4 NPs could catalyze the oxidation of typical per-
oxidase substrate 3,3 ,5,5 -tetramethylbenzidine (TMB) by H2 O2 to produce a visible blue color reaction
Keywords:
with absorption maxima centered at 652 nm. The CuWO4 NPs exhibit superior kinetics over the natu-
Peroxidase mimic
Spectrophotometry
ral enzyme horse radish peroxidase (HRP) with lower Km value. The CuWO4 NPs peroxidase mimicking
NADH sensor system was optimized for NADH sensing in the concentration range of 0.2–380 ␮M (R2 = 0.98) with a
Amperometry detection limit of 0.2 ␮M. Moreover, the CuWO4 NPs modified glassy carbon electrode (GCE) reduced
H2 O2 sensor H2 O2 at a lower onsite potential (−200 mV) thereby, allowing us to electrochemically sense hydrogen
peroxide with a linear range of 24.87 ␮M–3.6 mM (R2 = 0.99) and a sensitivity of 207.64 ␮A mM−1 .
© 2017 Elsevier B.V. All rights reserved.

1. Introduction been reported so far, to the best of our knowledge. Nicotinamide

Natural peroxidase enzymes, owing to their high efficiency
and substrate specificity, are extensively used in disease diag-
nosis, food processing, medicine, waste water treatment and
bio-sensing [1–5]. However, the expensive time-consuming sepa-
ration and purification techniques, large-scale dependency on pH,
temperature, and inhibitors makes natural enzymes unfavorable
for practical applications. Artificial peroxidase mimics, owing to
their tunable structures and chemical compositions coupled with
low cost, high stability, ease of preparation and superior properties
have attracted much interest [6,7]. Peroxidase mimics have been
successfully applied for the sensing of hydrogen peroxide [6,8], glu-
cose [8], cholesterol [6], cysteine [9], melamine [10], protein [11],
dopamine [12], sulfide [13], ascorbic acid [14], mercuric ions [15],
xanthine [16], acetamiprid [17], choline and acetylcholine [18],
uranyl ions [19], glyphosate [20], leukemia cancer cells [21] and
urea [22]. However, application of a peroxidase mimicking mate-
rial for nicotinamide adenine dinucleotide (NADH) sensing has not
∗ Corresponding author at: CSIR-Central Electrochemical Research Institute,
Karaikudi, 630003, Tamil Nadu, India.
E-mail addresses: sheelaberchmans@yahoo.com, sheelab@cecri.res.in
(S. Berchmans).
Adenine Dinucleotide (NADH) is one of the most important coen-
zymes found in human body. It can be formed in living cells through
the digestion process and more than 300 dehydrogenase enzymes
have been identified as NADH-dependent, maintaining the cellular
growth and energy metabolism [23–25]. NADH is directly linked to
the formation of adenosine triphosphate molecules (ATP), which is
regarded as the “molecular unit of currency” of intracellular energy
transfer and any deficiency of NADH will result in lower ATP pro-
duction and an energy deficit cellular level. Also, since, the brain
consumes one-third of energy produced in human cells, any deficit
in ATP can cause decreased mental alertness, attention deficit, pro-
longed reaction times, Alzheimer’s and dementia [26]. Low levels
NADH and the resulting decrease in the ATP may cause Parkin-
son’s disease because of the destruction of dopamine [27,28]. On
the other hand, increased concentration of NADH in the cellular
environment may lead to physiological effects such as anxiety,
insomnia, and fatigue [29]. Therefore, the development of a pre-
cise and selective sensor for measuring the concentration of NADH
in the presence of interfering biomolecules is extremely important.
Several methods such as electrochemiluminescence [30], liquid
chromatography [31], photoelectrocatalysis [32] etc are available
for the determination of NADH. However, these methods are often
time-consuming, lack sensitivity and are susceptible to interfer-
ence with other substances. Herein a simple colorimetric method

http://dx.doi.org/10.1016/j.snb.2017.06.175
0925-4005/© 2017 Elsevier B.V. All rights reserved.
724 A. K. et al. / Sensors and Actuators B 253 (2017) 723–730
for the selective determination of NADH is reported based on the
inhibition of peroxidase-like properties of CuWO4 NPs.
Tungstate, because of its excellent catalytic and electrochem-
ical characteristics has drawn wide attention. Metal tungstates
deliver good conductivity, as a result of the enhancement of the
electrical conductivity by W atoms (10−7 –10−3 S cm−1 ) and pro-
vide excellent electrochemical performance [33,34]. Further, metal
tungstates are applied for various usages in science and technol-
ogy such as radioactive decay of isotopes with very long half-life,
detection of non-baryonic dark matter double beta decay, solenoid
materials for specific calorimeters, in positron emission tomogra-
phy and as positive electrodes in lithium rechargeable batteries
[35–39]. Meanwhile, the 3d orbitals of the transition metals are
responsible for some of the electronic correlation effects observed
in these materials [40]. Among tungstates, copper tungstate, a well-
known n-type semiconductor with a bandgap of ca. 2.25–2.45 eV
and a member of the structurally related divalent transition metal
tungstates family, belongs to the distorted triclinic wolframite
structure [41–43]. In Copper tungstate, the constituent metal
atoms (Cu and W) are surrounded by six oxygen atoms. The
M–O distances within the CuO6 and WO6 octahedra fall in the
range of 0.1961–0.2450 nm and 0.1760–0.2208 nm respectively
[41]. This compound exhibits low dimensional magnetic behavior
as a result of the lightly coupled zigzag chains of copper ions. Many
applications of copper tungstate such as electrode material for
photoelectrolysis, photoanodes, optical fibers, solar-assisted water
splitting, laser host, scintillation detectors and sensors [44–53]
have been reported. However, the peroxidase mimetic properties
of CuWO4 NPs have not been reported so far to the best of our
knowledge.
2. Experimental
2.1. Materials
Hydrated sodium tungstate (Na2 WO4 .2H2 O), l-Ascorbic acid,
l-Cysteine, Glutathione, Copper (II) nitrate trihydrate, sodium
acetate, D(+)Glucose, acetic acid (glacial) and 3, 3 , 5, 5 -
tetramethylbenzidine (TMB) were purchased from Sigma-Aldrich.
Ethanol (99.8%) and Hydrogen peroxide (30%) were purchased
from Merck and ␤-Nicotinamide adenine dinucleotide, reduced
disodium salt hydrate (NADH) and uric acid were obtained from
acros organics. All chemicals were of highest purity available and
were used without further purification. Ultrapure Milli-Q water
with a resistance not less than 18.6 M was used throughout the
experiments.
2.2. Synthesis of CuWO4 NPs
The CuWO4 NPs were synthesized via chemical precipitation
reaction. In a typical experimental procedure, 2 mmol solutions of
Cu(NO3 )2 and Na2 WO4 were prepared separately in 25 mL of dis-
tilled water with stirring. The Na2 WO4 solution was then added
drop wise into Cu(NO3 )2 solution with frequent stirring to form a
cream green colored precipitate. After completion of the precipita-
tion reaction, the resultant product was transferred to a 100 mL
Teflon-lined autoclave and heated at 175 ◦ C for 24 h and then
allowed to cool to room temperature. The obtained precipitate was
then collected by centrifugation, washed with deionized water and
ethanol for several times and dried overnight. Finally, the samples
were calcinated at 300 ◦ C.
2.3. Apparatus and characterization
The surface morphology of the CuWO4 NPs was investigated
using a Tescan VEGA3 scanning electron microscope (SEM) and
Tecnai 20 G2 (FEI make) High-Resolution Transmission electron
microscope (HR-TEM). FT-IR results were obtained from a Bruker
Tensor 27, FT-IR Spectrometer. Kinetic studies, fluorescent exper-
iments, and UV–vis spectral measurements were performed on a
Tecan Infinite M200 pro spectrophotometer. The XPS analysis was
performed on a Multilab 2000 Base system with Mg/Al twin anode
(300/400W) X-Ray Source. XRD patterns were obtained from a
Bruker D8 Advance X-ray diffraction instrument using Ni-filtered
Cu K␣ radiation (l = 1.54 Å).
3. Results and discussion
3.1. Peroxidase-like activity
The peroxidase-like activities of the CuWO4 NPs were exam-
ined using H2 O2 and TMB substrates in acetate buffer solution
(1 mL, 0.1 M, pH = 3). The reaction was monitored on a Tecan Infi-
nite M200 pro spectrophotometer using the time drive mode. From
the initial linear region of the kinetic curves, the steady-state reac-
tion rates were obtained assuming molar absorption coefficient
of 39,000 M−1 cm−1 for TMB oxidation products at 652 nm. These
reaction rates were then fitted into the Michaelis–Menten (Eq. (1))
and Lineweaver-Burk (Eq. (2)) equations to calculate the kinetic
constants.
[S]
␯0 = ␯max (1)
[S] + Km
1 1 Km 1
= + × (2)
␯0 ␯max ␯max [S]
Where 0 is the reaction velocity, max is the maximal reaction
velocity, [S] is the substrate concentration (H2 O2 or TMB) and Km
is the Michaelis-Menten constant.
3.2. Electrochemistry experiments
The glassy carbon electrodes (GCE, 3.0 mm dia.) were first pol-
ished with 0.3 and 0.05 ␮m alumina slurry and then cleaned
in Milli-Q water by ultrasonication. 0.5 mg of CuWO4 NPs were
dispersed ultrasonically into 100 ␮L Milli-Q water to obtain a sus-
pension (5 mg ml−1 ). 10 ␮L of this suspension was then drop-cast
on the pretreated GCE and allowed to dry at room temperature
for one hour. All the electrochemical experiments were performed
on an AUTOLAB PGSTAT 302N work station. A three electrode sys-
tem comprising of CuWO4 NPs modified GCE (CuWO4 NPs/GCE)
as working electrode, Ag/AgCl as reference electrode and a spiral
platinum wire as auxiliary electrode was used in all electrochemical
studies. The experimental solutions were de-aerated by bubbling
nitrogen gas and were maintained under nitrogen atmosphere dur-
ing the electrochemical experiments.
Fig. 1a shows the FT-IR spectra of as-prepared CuWO4 NPs.
The peak around 3395 cm−1 is due to the stretching of O H and
bending of H O H bonds of the adsorbed water molecules on
the sample surface [51,54] and the peak appearing at 1608 cm−1
is associated with ı(OH) in W OH. The bands appearing between
880 and 470 cm−1 represent the wolframite structure. The absorp-
tion bands appearing below 600 cm−1 represent the deformation
modes of W O W bridges or W O bonds in the WO4 tetrahedra
[55]. Similarly, the stretching mode of W O bonds in junction with
WO4 tetrahedra appear at 890 cm−1 . Furthermore, the band with a
maxima of 770 cm−1 is due to Cu–O stretching [56].
To identify the crystal structure, phase, and lattice parameters,
the synthesized CuWO4 NPs were subjected to X-Ray Diffraction
analysis. Fig. 1b shows the XRD pattern of the synthesized cop-
per tungstate nanoparticles. The high crystallinity of the sample
is evident from the strong intensity peaks. Moreover, the diffrac-
tion peaks are in agreement with the wolframite structure of the
 A. K. et al. / Sensors and Actuators B 253 (2017) 723–730
Fig. 1. (a) FT-IR spectrum, (b) XRD pattern of the as-prepared CuWO4 NPs.
copper tungstate (Reference code: 01-073-1823) with space group
of P1 (2). The lattice parameters of the synthesized CuWO4 NPs
are a = 4.7026 Å, b = 5.8389 Å and c = 4.8784 Å with ␣ = 91.6770◦ ,
␤ = 92.4690◦ and ␥ = 82.8050◦ .
Then, as prepared CuWO4 NPs were characterized by SEM and
HR-TEM for determining their morphology. As shown in Fig. S1a
& 1b and Fig. 2a & b, the low and high-resolution SEM and HR-
TEM images revealed spherical morphology for CuWO4 NPs with an
average particle size of 66 nm. Simultaneously, the HRTEM images
(Fig. S1c & 1d, Fig. 2c) also showed visible lattice fringes for CuWO4
NPs with interplanar spacing of 0.296 nm, 0.210 nm, 0.186 nm,
0.225 nm, 0.251 nm, 0.243 nm, 0.171 nm, 0.201 nm, 0.155 nm and
0.579, which are in agreement with the interplanar spacing of the
(111), (−121), (130) (210), (0–21), (002), (−130), (211), (300) and
(010) facets of triclinic CuWO4 NPs. The dispersion of Copper, Tung-
sten, and Oxygen atoms in the catalyst was analyzed by STEM
elemental mapping. The elemental mapping (Fig. S2) confirmed the
presence of Cu, W and O atoms. In addition, the elemental map-
ping result also revealed the homogeneous existence of Cu, W and
O species.
XPS was used to identify the chemical state and surface com-
position of the catalyst. The survey spectrum of CuWO4 NPs, as
shown in Fig. 3a, indicates the presence of Cu 2p, O 1s, W 4f and
W 4d which are in good agreement with the EDAX results (Fig.
S3). The core-level spectrum of Cu 2p (Fig. 3b) shows peaks cor-
responding to Cu 2p3/2 and Cu 2p1/2 . The satellite peaks in the
spectra indicated that copper exists as Cu(II)[57]. The higher bind-
ing energy of Cu 2p3/2 than that of pure CuO (933.8 eV) suggests that
the Cu(II) species are surrounded by W-O. The core-level spectra of
W 4f5/2 and 4f7/2 (Fig. 3c) with the corresponding binding energies
37.1 eV and 34.8 eV suggest that the tungsten in the sample exists
as W+6 [58]. The core-level spectrum of O 1s (Fig. 3d) is decon-
voluted into three peaks, corresponding to the surface hydroxide
(O H, 531.6 eV) due to absorbed water, Cu-O groups (530.1 eV) and
W-O species (529.3 eV) [59].
3.3. Peroxidase-like properties of CuWO4 NPs
The peroxidase can catalyze the oxidation of some organic sub-
strates such as amines and phenols in presence of H2 O2 [60]. The
peroxidase-like activities of CuWO4 NPs were investigated by the
oxidation of TMB in the presence of H2 O2 . As shown in Fig. S4, in the
absence of CuWO4 NPs catalyst H2 O2 can’t oxidize TMB similar to
the case reported for horseradish peroxidase enzyme [61], and with
the addition of CuWO4 NPs catalyst, the oxidation of TMB devel-
oped characteristic blue color with absorbance maxima centered at
652 nm. The dependency of external conditions on the peroxidase-
like activity of CuWO4 NPs was investigated by varying the pH and
725
temperature respectively. As shown in Fig. S5, the catalytic activity
of CuWO4 NPs is dependent on pH and temperature. The optimal
pH for maximum catalytic activity was observed at pH 3 and the
catalytic activity continued to increase with increase in tempera-
ture reaching maximum at 80 ◦ C. This behavior resembles many
reported peroxidase mimicking materials [62,63] and indicates the
robustness of the catalyst under harsh conditions of temperature
and pH.
3.4. Kinetics of the peroxidase- like property and proposed
mechanism
The peroxidase-like properties of CuWO4 NPs were further
investigated by steady-state kinetic analysis (Fig. S6). The kinet-
ics was measured by keeping the concentration of one of the
peroxidase substrate (TMB or H2 O2 ) constant while varying the
concentration of other (Fig. S7). Michaelis–Menten curves for
CuWO4 NPs (Fig. S7a & 7b) were obtained from the initial kinetic
curves. Lineweaver–Burk plots (double reciprocal plots) were then
plotted as the double reciprocal using three different concentra-
tions of TMB and showed good linear relationship with parallel
lines (Fig. S7c). These parallel lines are characteristic of a ping-pong
mechanism similar to that of the natural horse radish peroxidase
enzyme [61]. Lineweaver–Burk plots (Fig. S7c) were then plot-
ted as the double reciprocal and showed good linear relationship.
These results confirmed typical Michaelis–Menten model for the
peroxidase reaction catalyzed by CuWO4 NPs. A comparison of
the kinetic parameters obtained from the Michaelis–Menten and
Lineweaver–Burk plots with recent literature reports are shown
in Table 1. The Km values are directly related to the affinity of
an enzyme towards the substrates, and smaller Km values indi-
cated higher affinity between the enzyme and the substrates and
vice versa. The observed Km (0.385 mM) value of CuWO4 NPs with
H2 O2 substrate is found to be smaller than that of the natural horse
radish peroxidase enzyme, suggesting the higher affinity of the cat-
alyst for H2 O2 . The availability of more active sites on the CuWO4
NPs surface may be the reason for the lower Km value than the
natural horse radish peroxidase, which has only one active site per
molecule. The ␯max for the CuWO4 NPs, which is the maximum
reaction velocity when the enzyme is saturated with the substrate
was found to be higher than HRP indicating the higher catalytic
activity.
In order to further investigate the mechanism of peroxidase-
like activity, the electrochemical behavior of the CuWO4 NPs was
analyzed using cyclic voltammetry and amperometry. As shown
in Fig. 4a, no obvious current was observed in the blank experi-
ment, however, with the addition of 0.49 mM H2 O2 , the reduction
current increased steadily indicating the ability of CuWO4 NPs to
726 A. K. et al. / Sensors and Actuators B 253 (2017) 723–730

Fig. 2. (a–c) HR-TEM images of CuWO4 NPs under different resolutions (d) the corresponding SAED pattern.

Fig. 3. (a) XPS survey scan of CuWO4 NPs, (b) Core-level spectrum of Cu 2p, (c) Core-level spectrum of W, (d) O 1s core-level spectrum.
 A. K. et al. / Sensors and Actuators B 253 (2017) 723–730 727

Table 1
Comparison of the kinetic parameters with recent literature value.

Catalysts Km (mM) ␯max [10−8 × Ms−1 ] References

H2 O2 TMB H2 O2 TMB

HRP 7.08 0.12 491 14.5 [64]

TemFe A 157.19 0.08 1.28 0.97 [65]
Ni/Co LDHs 13.2 – 3.24 – [66]
Fe3 O4 MNPs 154 0.09 9.78 3.44 [61]
Cu-Ag/rGO 8.62 0.63 7.01 4.25 [67]
Nanosized CuS 1.75 0.06 23.7 76.4 [68]
Fe2 (MoO4 )3 -F 0.10 1.12 7.51 3.73 [69]
Pd-Ir cubes 340 0.13 5.1 6.5 [70]
Fluorescein 1.11 1.31 0.43 0.41 [71]
Co3 O4 @CeO2 7.09 0.14 4.33 4.13 [72]
CuWO4 NPs 0.38 0.20 472 11.3 This work

Fig. 4. a) Cyclic voltammograms obtained at the GCE/CuWO4 electrode (␸ = 3 mm) at a scan rate of 50 mVs−1 in acetate buffer solution (0.1 M, pH = 3) in the absence and in
the presence of different concentrations of H2 O2 . b) Amperometric response of the GCE/CuWO4 for the continuous additions of H2 O2 at applied potential of −0.2 V.

Scheme 1. Proposed NADH sensing mechanism using the peroxidase mimicking properties of CuWO4 NPs.

transfer electrons between electrode surface (electron donor) and rent increased steadily with a high sensitivity of 207.64 ␮A mM−1
H2 O2 (electron acceptor). The amperometric response is recorded along with each addition of H2 O2 . These results indicated that the
at an applied potential of −200 mV (Fig. 4b). The reduction cur- CuWO4 NPs fulfill the electron transfer between H2 O2 and TMB and
728 A. K. et al. / Sensors and Actuators B 253 (2017) 723–730

Fig. 5. (a) Digital images of the CuWO4 NPs system in the absence and presence of different concentrations of NADH. From left to right, the concentration of NADH are 0,
0.2, 0.38, 2, 3.8, 20, 40, 77, 100, 200, 380 and 555 ␮M, respectively, (b) UV–vis spectra of the proposed system in the presence of different concentrations of NADH, (c) Linear
calibration plot for NADH detection. The error bars indicate standard deviation for n=3.

exhibit the intrinsic peroxidase-like activity. Therefore, the pro-

posed mechanism is as follows (Scheme 1). TMB after adsorbing
on the surface of CuWO4 NPs due to the ␲–␲ stacking interaction
[73], donates a lone-pair of electrons from the amino groups to the
CuWO4 NPs, thereby increasing the electron density and mobility in
CuWO4 NPs. This process also oxidizes TMB into TMB.+ with char-
acteristic absorbance maxima at 652 nm. The electron rich catalyst
center then transfers the electrons to H2 O2 , thereby reducing it to
water [74]. Besides, the large specific surface area and small crys-
talline size of CuWO4 NPs may provide high density of catalytically
active sites that bind substrates with less steric hindrance and more
powerful redox capability because of the quantum-size effect [75].

3.5. Analytical performance of the proposed sensor

Interestingly, it was found that nicotinamide adenine dinucleo-

tide (NADH) could inhibit the peroxidase-like properties of CuWO4
NPs. The finding above led us to develop a NADH sensor. The perfor-
mance of the proposed sensor was evaluated by recording the color
changes of CuWO4 NPs system in the absence and presence of dif-
ferent amounts of NADH. As shown in Fig. 5a, a clear visual change
in the blue color is observed which could be differentiated with
the naked eyes. The catalytic conditions were then optimized for
the optimal detection of NADH. The UV–vis spectra of the CuWO4
NPs system in the presence of different concentrations of NADH
are shown in Fig. 5b. It can be seen that the absorbance at 652 nm
decreased gradually with the increase in NADH concentration. The
NADH sensor exhibited a good linear relationship in the concentra-
tion range from 0.2 ␮M–380 ␮M with a detection limit of 0.2 ␮M.
As shown in Table.S1 (Supporting information), a comparison of the
analytical parameters with recent literature reports shows that the
sensor is superior to many of the reported methods. The repeatabil-
ity studies performed using five independent measurements with
40 ␮M NADH shows a relative standard deviation (RSD) of 3.9%,
showing the reliability of the proposed sensor.
Fig. 6. (a) Typical UV–vis spectra of 50 ␮g ml−1 of CuWO4 NPs in the presence of
0.24 mM TMB and 4.7 mM H2 O2 , (b) 50 ␮g ml−1 of CuWO4 NPs in the presence of
0.24 mM TMB, 4.7 mM H2 O2 and 0.45 mM NADH, (c) 50 ␮g ml−1 of CuWO4 NPs in the
presence of 0.24 mM TMB, 4.7 mM H2 O2 and 0.45 mM glutathione, (d) 50 ␮g ml−1
of CuWO4 NPs in the presence of 0.24 mM TMB, 4.7 mM H2 O2 and 0.45 mM cystein,
(e) 50 ␮g ml−1 of CuWO4 NPs in the presence of 0.24 mM TMB, 4.7 mM H2 O2 and
0.45 mM ascorbic acid, (f) 50 ␮g ml−1 of CuWO4 NPs in the presence of 0.24 mM TMB,
4.7 mM H2 O2 and 0.45 mM uric acid, (g) 50 ␮g ml−1 of CuWO4 NPs in the presence of
0.24 mM TMB, 4.7 mM H2 O2 and 0.45 mM dopamine. (Inset shows the digital images
of the corresponding samples).
3.6. Selectivity studies
The selectivity of the proposed sensing platform was investi-
gated with potential interfering substances including glutathione,
cysteine, ascorbic acid, uric acid, glucose, and dopamine. As shown
in Fig. 6 & Fig. S8, it is clearly seen that only NADH induces a dra-
matic decrease in the peroxidase- like properties of CuWO4 NPs
when compared to other interfering substances, suggesting that the
 A. K. et al. / Sensors and Actuators B 253 (2017) 723–730
proposed method is appropriate for the highly selective detection
of NADH.
4. Conclusions
The CuWO4 NPs were found to exhibit properties similar to that
of peroxidase enzyme. The obtained Km value of the CuWO4 NPs
were found to be lower than the natural enzyme horse radish per-
oxidase (HRP) and many recently reported peroxidase mimicking
materials. The peroxidase inhibition effect of NADH over CuWO4
NPs was exploited to develop a highly selective NADH sensing plat-
form. The peroxidase mimicking system was optimized to fabricate
a NADH sensor in the concentration range of 0.2- 380 ␮M (R2 = 0.98)
with a detection limit of 0.2 ␮M. Moreover, the lower onsite reduc-
tion potential (–200 mV) of H2 O2 over the CuWO4 NPs modified
glassy carbon electrode (GCE/CuWO4 ) was applied for the fabrica-
tion of an amperometric hydrogen peroxide sensor with a linear
range of 24.87 ␮M–3.6 mM and a sensitivity of 207.64 ␮A mM−1 .
Acknowledgements
One of the authors K. A is thankful to CSIR, New Delhi for pro-
viding senior research fellowship. The authors also acknowledge
the CSIR five-year plan M2D (Grant number CSC-0134) for provid-
ing the necessary funding. Central Instrumentation Facility of CSIR-
CECRI is also greatly appreciated for the necessary characteriza-
tions.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.snb.2017.06.175.
References
[1] F. Burnette, Peroxidase and its relationship to food flavor and quality: a
review, J. Food Sci. 42 (1977) 1–6.
[2] J. Czachurski, K. Lackner, D. Ockert, H. Seller, Localization of neurones with
baroreceptor input in the medial solitary nucleus by means of intracellular
application of horseradish peroxidase in the cat, Neurosci. Lett. 28 (1982)
133–137.
[3] W.D. Geoghegan, G.A. Ackerman, Adsorption of horseradish peroxidase,
ovomucoid and anti-immunoglobulin to colloidal gold for the indirect
detection of concanavalin A, wheat germ agglutinin and goat anti-human
immunoglobulin G on cell surfaces at the electron microscopic level: a new
method, theory and application, J. Histochem. Cytochem. 25 (1977)
1187–1200.
[4] A.M. Klibanov, E.D. Morris, Horseradish peroxidase for the removal of
carcinogenic aromatic amines from water, Enzyme Microb. Technol. 3 (1981)
119–122.
[5] A.C. Patel, S. Li, J.-M. Yuan, Y. Wei, In situ encapsulation of horseradish
peroxidase in electrospun porous silica fibers for potential biosensor
applications, Nano Lett. 6 (2006) 1042–1046.
[6] K. Aneesh, C.S.R. Vusa, S. Berchmans, Impurity-induced peroxidase mimicry of
nanoclay and its potential for the spectrophotometric determination of
cholesterol, Anal. Bioanal. Chem. 408 (2016) 6213–6221.
[7] Y. Tao, Y. Lin, Z. Huang, J. Ren, X. Qu, Incorporating graphene oxide and gold
nanoclusters: a synergistic catalyst with surprisingly high peroxidase-like
activity over a broad pH range and its application for cancer cell detection,
Adv. Mater. 25 (2013) 2594–2599.
[8] K. Aneesh, C.S.R. Vusa, S. Berchmans, Dual enzyme mimicry exhibited by ITO
nanocubes and their application in spectrophotometric and electrochemical
sensing, Analyst 141 (2016) 4024–4028.
[9] R.S. Li, H. Liu, B.B. Chen, H.Z. Zhang, C.Z. Huang, J. Wang, Stable gold
nanoparticles as a novel peroxidase mimic for colorimetric detection of
cysteine, Anal. Methods 8 (2016) 2494–2501.
[10] P. Ni, H. Dai, Y. Wang, Y. Sun, Y. Shi, J. Hu, et al., Visual detection of melamine
based on the peroxidase-like activity enhancement of bare gold
nanoparticles, Biosens. Bioelectron. 60 (2014) 286–291.
[11] S. Kumari, B.B. Dhar, C. Panda, A. Meena, S. Sen Gupta, Fe-TAML encapsulated
inside mesoporous silica nanoparticles as peroxidase mimic: femtomolar
protein detection, ACS Appl. Mater. Interfaces 6 (2014) 13866–13873.
[12] S. Dutta, C. Ray, S. Mallick, S. Sarkar, R. Sahoo, Y. Negishi, et al., A gel-based
approach to design hierarchical CuS decorated reduced graphene oxide
729
nanosheets for enhanced peroxidase-like activity leading to colorimetric
detection of dopamine, J. Phys. Chem. C 119 (2015) 23790–23800.
[13] S. Singh, K. Mitra, A. Shukla, R. Singh, R.K. Gundampati, N. Misra, et al.,
Brominated graphene as mimetic peroxidase for sulfide ion recognition, Anal.
Chem. 89 (2017) 783–791.
[14] J. Chen, J. Ge, L. Zhang, Z. Li, J. Li, Y. Sun, et al., Reduced graphene oxide
nanosheets functionalized with poly(styrene sulfonate) as a peroxidase
mimetic in a colorimetric assay for ascorbic acid, Microchim. Acta 183 (2016)
1847–1853.
[15] W. Li, B. Chen, H. Zhang, Y. Sun, J. Wang, J. Zhang, et al., BSA-stabilized Pt
nanozyme for peroxidase mimetics and its application on colorimetric
detection of mercury (II) ions, Biosens. Bioelectron. 66 (2015) 251–258.
[16] M. Cui, J. Zhou, Y. Zhao, Q. Song, Facile synthesis of iridium nanoparticles with
superior peroxidase-like activity for colorimetric determination of H2O2 and
xanthine, Sens. Actuators B: Chem. 243 (2017) 203–210.
[17] Z. Yang, J. Qian, X. Yang, D. Jiang, X. Du, K. Wang, et al., A facile label-free
colorimetric aptasensor for acetamiprid based on the peroxidase-like activity
of hemin-functionalized reduced graphene oxide, Biosens. Bioelectron. 65
(2015) 39–46.
[18] S.-B. He, G.-W. Wu, H.-H. Deng, A.-L. Liu, X.-H. Lin, X.-H. Xia, et al., Choline and
acetylcholine detection based on peroxidase-like activity and protein
antifouling property of platinum nanoparticles in bovine serum albumin
scaffold, Biosens. Bioelectron. 62 (2014) 331–336.
[19] D. Zhang, Z. Chen, H. Omar, L. Deng, N.M. Khashab, Colorimetric peroxidase
mimetic assay for uranyl detection in sea water, ACS Appl. Mater. Interfaces 7
(2015) 4589–4594.
[20] Y. Chang, Z. Zhang, J. Hao, W. Yang, J. Tang, A simple label free colorimetric
method for glyphosate detection based on the inhibition of peroxidase-like
activity of Cu(II), Sens. Actuators B: Chem. 228 (2016) 410–415.
[21] J. Liu, M. Cui, L. Niu, H. Zhou, S. Zhang, Enhanced peroxidase-like properties of
graphene–hemin-composite decorated with Au nanoflowers as
electrochemical aptamer biosensor for the detection of K562 leukemia cancer
cells, Chem. – A Eur. J. 22 (2016) 18001–18008.
[22] H.-H. Deng, G.-L. Hong, F.-L. Lin, A.-L. Liu, X.-H. Xia, W. Chen, Colorimetric
detection of urea, urease, and urease inhibitor based on the peroxidase-like
activity of gold nanoparticles, Anal. Chim. Acta 915 (2016) 74–80.
[23] M.I. Álvarez-González, S.B. Saidman, M.J. Lobo-Castañón, A.J.
Miranda-Ordieres, P. Tuñón-Blanco, Electrocatalytic detection of NADH and
glycerol by NAD+-modified carbon electrodes, Anal. Chem. 72 (2000)
520–527.
[24] L. Gorton, E. Domıı́, Electrocatalytic oxidation of NAD (P) H at
mediator-modified electrodes, Rev. Mol. Biotechnol. 82 (2002) 371–392.
[25] L. Tang, G.-M. Zeng, H. Wang, G.-L. Shen, D.-L. Huang, Amperometric detection
of lignin-degrading peroxidase activities from Phanerochaetechrysosporium,
Enzyme Microb. Technol. 36 (2005) 960–966.
[26] F.S. Omar, N. Duraisamy, K. Ramesh, S. Ramesh, Conducting polymer and its
composite materials based electrochemical sensor for Nicotinamide Adenine
Dinucleotide (NADH), Biosens. Bioelectron. 79 (2016) 763–775.
[27] J. Castro-Marrero, M.D. Cordero, M.J. Segundo, N. Sáez-Francàs, N. Calvo, L.
Román-Malo, et al., Does oral coenzyme Q(10) plus NADH supplementation
improve fatigue and biochemical parameters in chronic fatigue syndrome?
Antioxid. Redox Signaling 22 (2015) 679–685.
[28] G.D. Birkmayer, NADH, the Biological Hydrogen: The Secret of Our Life
Energy, Basic Health Publications, Inc., 2009.
[29] R. Sahelian, NADH Supplement Health Benefit, Side Effects 5, 2016, URL:
<http://www.raysahelian.com/nadh.html> (accessed on 17.05.17).
[30] Y. Li, X. Yang, F. Yang, Y. Wang, P. Zheng, X. Liu, Effective immobilization of
Ru(bpy)32+ by functional composite phosphomolybdic acid anion on an
electrode surface for solid-state electrochemiluminescene to sensitive
determination of NADH, Electrochim. Acta 66 (2012) 188–192.
[31] C. Brugidou, A. Rocher, E. Giraud, B. Lelong, B. Marin, M. Raimbault, A new
high performance liquid chromatographic technique for separation and
determination of adenylic and nicotinamide nucleotides in Lactobacillus
plantarum, Biotechnol. Tech. 5 (1991) 475–478.
[32] D.G. Dilgin, D. Gligor, H.İ. Gökçel, Z. Dursun, Y. Dilgin, Photoelectrocatalytic
oxidation of NADH in a flow injection analysis system using a
poly-hematoxylin modified glassy carbon electrode, Biosens. Bioelectron. 26
(2010) 411–417.
[33] D. Hu, P. Diao, D. Xu, M. Xia, Y. Gu, Q. Wu, et al., Copper (II) tungstate
nanoflake array films: sacrificial template synthesis, hydrogen treatment, and
their application as photoanodes in solar water splitting, Nanoscale 8 (2016)
5892–5901.
[34] F. Zhan, J. Li, W. Li, Y. Liu, R. Xie, Y. Yang, et al., In situ formation of CuWO 4/WO
3 heterojunction plates array films with enhanced photoelectrochemical
properties, Int. J. Hydrogen Energy 40 (2015) 6512–6520.
[35] F. Bonino, B. Di Pietro, B. Rivolta, B. Scrosati, Lithium-copper molybdate and
lithium-copper Tungstate organic solvent batteries, J. Power Sources 2 (1978)
265–272.
[36] M. Kobayashi, Y. Usuki, M. Ishii, M. Itoh, M. Nikl, Further study on different
dopings into PbWO 4 single crystals to increase the scintillation light yield,
Nucl. Instrum. Methods Phys. Res. Sect. A 540 (2005) 381–394.
[37] M. Lalić, Z. Popović, F. Vukajlović, Ab initio study of electronic, magnetic and
optical properties of CuWO 4 tungstate, Comput. Mater. Sci 50 (2011)
1179–1186.
[38] V.B. Mikhailik, H. Kraus, Cryogenic scintillators in searches for extremely rare
events, J. Phys. D: Appl. Phys. 39 (2006) 1181.
730 A. K. et al. / Sensors and Actuators B 253 (2017) 723–730
[39] M. Nikl, P. Bohacek, E. Mihokova, M. Kobayashi, M. Ishii, Y. Usuki, et al.,
Excitonic emission of scheelite tungstates AWO 4 (A = Pb, Ca, Ba, Sr), J. Lumin.
87 (2000) 1136–1139.
[40] S. Arora, T. Mathew, B. Chudasama, A. Kothari, Single crystal growth and
photoelectrochemical study of copper tungstate, J. Cryst. Growth 275 (2005)
e651–e666.
[41] L. Kihlborg, E. Gebert, CuWO4, a distorted wolframite-type structure, Acta
Crystallogr. Sect. B: Struct. Crystallogr. Cryst. Chem. 26 (1970) 1020–1026.
[42] S. Klein, H. Weitzel, PERNOD-A program for refinement of crystal structure
parameters from neutron powder diffraction diagrams, J. Appl. Crystallogr. 8
(1975) 54–59.
[43] P.F. Schofield, K. Knight, S. Redfern, G. Cressey, Distortion characteristics
across the structural phase transition in (Cu1- xZnx) WO4, Acta Crystallogr.
Sect. B: Struct. Sci. 53 (1997) 102–112.
[44] N. Gaillard, Y. Chang, A. DeAngelis, S. Higgins, A. Braun, A nanocomposite
photoelectrode made of 2.2 eV band gap copper tungstate (CuWO 4) and
multi-wall carbon nanotubes for solar-assisted water splitting, Int. J.
Hydrogen Energy 38 (2013) 3166–3176.
[45] R. Gillette, Calcium and cadmium tungstate as scintillation counter crystals
for gamma-ray detection, Rev. Sci. Instrum. 21 (1950) 294–301.
[46] C.R. Lhermitte, B.M. Bartlett, Advancing the chemistry of CuWO4 for
photoelectrochemical water oxidation, Acc. Chem. Res. 49 (2016) 1121–1129.
[47] C.-L. Li, Z.-W. Fu, Nano-sized copper tungstate thin films as positive electrodes
for rechargeable Li batteries, Electrochim. Acta 53 (2008) 4293–4301.
[48] P.K. Pandey, N. Bhave, R. Kharat, Spray deposition process of polycrystalline
thin films of CuWO 4 and study on its photovoltaic electrochemical
properties, Mater. Lett. 59 (2005) 3149–3155.
[49] M. Peter, Millimeter-wave paramagnetic resonance spectrum of S 6 state
impurity (Fe+++) in MgW O 4, Phys. Rev. 113 (1959) 801.
[50] S.M. Pourmortazavi, M. Rahimi-Nasrabadi, Y. Fazli, M. Mohammad-Zadeh,
Taguchi method assisted optimization of electrochemical synthesis and
structural characterization of copper tungstate nanoparticles, Int. J. Refract.
Met. Hard Mater. 51 (2015) 29–34.
[51] S.A. Redfern, Hard-mode infrared study of the ferroelastic phase transition in
CuWO 4-ZnWO4 mixed crystals, Phys. Rev. B 48 (1993) 5761.
[52] L. Van Uitert, S. Preziosi, Zinc tungstates for microwave maser applications, J.
Appl. Phys. 33 (1962) 2908–2909.
[53] X. Xu, J. Shen, N. Li, M. Ye, Facile synthesis of reduced graphene oxide/CoWO 4
nanocomposites with enhanced electrochemical performances for
supercapacitors, Electrochim. Acta 150 (2014) 23–34.
[54] O.Y. Khyzhun, V. Bekenev, Y.M. Solonin, First-principles calculations and
X-ray spectroscopy studies of the electronic structure of CuWO 4, J. Alloys
Compd. 480 (2009) 184–189.
[55] S. Naik, A. Salker, Solid state studies on cobalt and copper tungstates nano
materials, Solid State Sci. 12 (2010) 2065–2072.
[56] S.M. Pourmortazavi, M. Rahimi-Nasrabadi, Y. Fazli, M. Mohammad-Zadeh,
Taguchi method assisted optimization of electrochemical synthesis and
structural characterization of copper tungstate nanoparticles, Int. J. Refract.
Met. Hard Mater. 51 (2015) 29–34.
[57] S.S. Acharyya, S. Ghosh, R. Bal, Nanoclusters of Cu (II) supported on
nanocrystalline W (VI) oxide: a potential catalyst for single-step conversion of
cyclohexane to adipic acid, Green Chem. 17 (2015) 3490–3499.
[58] S.A.K. Leghari, S. Sajjad, J. Zhang, A time saving and cost effective route for
metal oxides activation, RSC Adv. 4 (2014) 5248–5253.
[59] C. Wei, Y. Huang, X. Zhang, X. Chen, J. Yan, Soft-template hydrothermal
systhesis of nanostructured Copper (II) tungstate cubes for electrochemical
charge storage application, Electrochim. Acta 220 (2016) 156–163.
[60] M.O. Senge, S.A. MacGowan, J.M. O’Brien, Conformational control of cofactors
in nature–the influence of protein-induced macrocycle distortion on the
biological function of tetrapyrroles, Chem. Commun. 51 (2015) 17031–17063.
[61] L. Gao, J. Zhuang, L. Nie, J. Zhang, Y. Zhang, N. Gu, et al., Intrinsic
peroxidase-like activity of ferromagnetic nanoparticles, Nat. Nanotechnol. 2
(2007) 577–583.
[62] H. Liu, C. Gu, W. Xiong, M. Zhang, A sensitive hydrogen peroxide biosensor
using ultra-small CuInS 2 nanocrystals as peroxidase mimics, Sens. Actuators
B: Chem. 209 (2015) 670–676.
[63] F. Qiao, L. Chen, X. Li, L. Li, S. Ai, Peroxidase-like activity of manganese selenide
nanoparticles and its analytical application for visual detection of hydrogen
peroxide and glucose, Sens. Actuators B: Chem. 193 (2014) 255–262.
[64] L. Zhang, L. Han, P. Hu, L. Wang, S. Dong, TiO2 nanotube arrays: intrinsic
peroxidase mimetics, Chem. Commun. 49 (2013) 10480–10482.
[65] A. Roy, R. Sahoo, C. Ray, S. Dutta, T. Pal, Soft template induced phase selective
synthesis of Fe2O3 nanomagnets: one step towards peroxidase-mimic
activity allowing colorimetric sensing of thioglycolic acid, RSC Adv. 6 (2016)
32308–32318.
[66] L. Su, X. Yu, W. Qin, W. Dong, C. Wu, Y. Zhang, et al., One-step analysis of
glucose and acetylcholine in water based on the intrinsic peroxidase-like
activity of Ni/Co LDHs microspheres, J. Mater. Chem. B 5 (2017) 116–122.
[67] G. Darabdhara, B. Sharma, M.R. Das, R. Boukherroub, S. Szunerits, Cu-Ag
bimetallic nanoparticles on reduced graphene oxide nanosheets as
peroxidase mimic for glucose and ascorbic acid detection, Sens. Actuators B:
Chem. 238 (2017) 842–851.
[68] X. Niu, Y. He, J. Pan, X. Li, F. Qiu, Y. Yan, et al., Uncapped nanobranch-based
CuS clews used as an efficient peroxidase mimic enable the visual detection of
hydrogen peroxide and glucose with fast response, Anal. Chim. Acta 947
(2016) 42–49.
[69] B. Wang, P. Ju, D. Zhang, X. Han, L. Zheng, X. Yin, et al., Colorimetric detection
of H2O2 using flower-like Fe2 (MoO4) 3 microparticles as a peroxidase
mimic, Microchim. Acta 183 (2016) 3025–3033.
[70] X. Xia, J. Zhang, N. Lu, M.J. Kim, K. Ghale, Y. Xu, et al., Pd–Ir core–shell
nanocubes: a type of highly efficient and versatile peroxidase mimic, ACS
Nano 9 (2015) 9994–10004.
[71] L. Liu, Y. Shi, Y. Yang, M. Li, Y. Long, Y. Huang, et al., Fluorescein as an artificial
enzyme to mimic peroxidase, Chem. Commun. 52 (2016) 13912–13915.
[72] D. Jampaiah, T. Srinivasa Reddy, V.E. Coyle, A. Nafady, S.K. Bhargava,
Co3O4@CeO2 hybrid flower-like microspheres: a strong synergistic
peroxidase-mimicking artificial enzyme with high sensitivity for glucose
detection, J. Mater. Chem. B 5 (2017) 720–730.
[73] J. Mu, J. Li, X. Zhao, E.-C. Yang, X.-J. Zhao, Cobalt-doped graphitic carbon
nitride with enhanced peroxidase-like activity for wastewater treatment, RSC
Adv. 6 (2016) 35568–35576.
[74] J. Tian, Q. Liu, A.M. Asiri, A.H. Qusti, A.O. Al-Youbi, X. Sun, Ultrathin graphitic
carbon nitride nanosheets: a novel peroxidase mimetic, Fe doping-mediated
catalytic performance enhancement and application to rapid, highly sensitive
optical detection of glucose, Nanoscale 5 (2013) 11604–11609.
[75] J.C. Yu, L. Zhang, J. Yu, Direct sonochemical preparation and characterization
of highly active mesoporous TiO2 with a bicrystalline framework, Chem.
Mater. 14 (2002) 4647–4653.
Biographies
Mr. K. Aneesh has received his B.Sc. degree in Chemistry from University of Calicut,
Kerala, India, in the year 2007 and M.Sc. degree in Chemistry from Mahatma Gandhi
University, Kerala, India, in the year 2009. After his Masters, he joined Ph.D. under
the supervision of Dr. Sheela Berchmans, Chief Scientist, Central Electrochemical
Research Institute. Currently, he is a Ph.D. student of Academy of Scientific and
Innovative Research (AcSIR), New Delhi, India. His research interests are related to
the electrochemical and spectrophotometric sensing applications of heterogeneous
enzyme mimics.
Dr. Sheela Berchmans is currently working as a Chief scientist in CSIR-Central Elec-
trochemical Research Institute, Karaikudi, Tamilnadu, India. She obtained her PhD
degree from The Alagappa University in 1993 and her research interests include
Biosensors, Microbial fuel cells, Electro catalysis, Bio and chemical synthesis of
nanomaterials and preparation of NO releasing materials.